Protocol for Analyzing of the Effect of Sphingomyelinase on Rubella Virus Infectivity in Two Cell Lines

Experiment Summary

Rubella is a mildly contagious disease characterized by low-grade fever and a morbilliform rash caused by the rubella virus (RuV). Viruses often use cellular phospholipids for infection. This protocol studied the roles of cellular sphingomyelin in RuV infection. Treatment of cells with sphingomyelinase (SMase) inhibited RuV infection in rabbit kidney-derived RK13 cells and African green monkey (Cercopithecus aethiops) kidney-derived Vero cells. RuV used cellular sphingomyelin and cholesterol for its binding to cells and membrane fusion at the step of virus entry. Detailed protocols of the assays, which assess the effects of SMase treatment on RuV infectivity in RK13 and Vero cells, are described.

Materials and Reagents

A. Materials

A 6-well cell culture plate
A 12-well cell culture plate
A 24-well cell culture plate
Pipette tips
A 1.5 ml tube with lid

B. Cells

RK13 cells: these cells were a gift from the Kitasato Institute
Vero cells: our pre-existing stocks were originally obtained from American Type Culture Collection and have been maintained for more than 20 years in our laboratory

C. Viruses

Rubella virus (RuV) TO-336 vaccine strain
RuV RVi/Hiroshima.JPN/01.03 wild type strain
The recombinant RuV RVi/Hiroshima.JPN/01.03 strain expressing the green fluorescent AG1 protein (rHS/p150-AG1)
The recombinant measles virus (MeV) and human metapneumovirus (HMPV) expressing enhanced green fluorescent protein (EGFP) (MeV-IC323/Ed-H-EGFP and HMPV-rJPS02-76EGFP, respectively)

D. Virus-like particles of RuV (RuV-VLP)

RuV-VLP, whose genome encodes Renilla luciferase and AG1 reporter genes, were produced and concentrated as described previously, with some modifications.

E. Reagents

Eagle's MEM "Nissui" 1
Dulbecco's modified Eagle's medium
Dulbecco's phosphate buffered saline (DPBS)
Fetal bovine serum
Bovine serum
Penicillin-streptomycin
L-Glutamine (200 mM)
Agarose ME
7.5% sodium bicarbonate
Sphingomyelinase from Bacillus cereus
Neutral red solution (0.33%)
Renilla Luciferase Assay system
MEM
2x MEM
MEM containing 2% BS (or FBS) and 0.5% (or 0.4%) agarose
MEM containing 0.01% neutral red and 0.5% agarose

Equipment

Micropipettes
Luminometer
Autoclave
Biosafety cabinet
Cell culture incubator (at 35 °C and 37 °C with 5% CO₂)
Fluorescence microscope
Cell culture microscope

Procedure

A. Analysis of the effect of SMase treatment on viral infectivity in RK13 cells (Figure 1A)

Figure 1. Experimental flow.

Seed 3 ml of a RK13 cell suspension at a concentration of 250,000 cells/ml into each well of a 6-well plate.
Incubate the cells in a cell culture incubator at 37 °C with 5% CO₂.
After cells form a monolayer (3-4 days post seeding), wash the cells with DPBS once, and then add 1 ml of MEM containing 30 or 150 mU/ml of SMase or 1 ml of MEM as a non-treated control.
Incubate the cells for 1 h in a cell culture incubator at 37 °C with 5% CO₂.
Wash the cells with DPBS twice. (Remove the DPBS after washing).
Add 0.1 ml of a RuV-containing solution at a concentration of 300-800 plaque-forming units/ml.
Incubate the cells with the virus solution for 1 h at room temperature with gentle agitation every 15 min.
Add 3 ml of MEM containing 2% BS and 0.5% agarose.
Incubate the cells for 7 days in a cell culture incubator at 35 °C with 5% CO₂.
Add 1 ml of MEM containing 0.01% neutral red and 0.5% agarose.
Incubate for 2 to 3 days in a cell culture incubator at 35 °C with 5% CO₂.
Count the number of plaques with the naked eye.

Figure 2. Effect of SMase treatment on RuV infection in RK13 cells.

B. Analysis of the effect of SMase treatment on viral infectivity in Vero cells (Figure 1B).

Seed 1 ml of a Vero cell suspension at a concentration of 300,000 cells/ml into each well of a 12-well plate.
Incubate the cells in a cell culture incubator at 37 °C with 5% CO₂.
After cells form a monolayer (16-36 h post seeding), wash the cells with DPBS once, and then add 0.5 ml of DMEM containing 30 or 150 mU/ml of SMase or 0.5 ml of DMEM as a non-treated control. (Remove the DPBS after washing).
Incubate the cells for 1 h in a cell culture incubator at 37 °C with 5% CO₂.
Wash the cells with DPBS twice. (Remove the DPBS after washing).
Add 0.1 ml of a virus solution containing RuV rHS/p150-AG1, MeV-IC323/Ed-H-EGFP, or HMPV-rJPS02-76EGFP at a concentration of 300-1000 fluorescent focus-forming units/ml.
Incubate the cells with the virus solution for 1 h at room temperature with gentle agitation every 20 min.
Remove the virus solution, wash the cells with MEM twice to remove unbound viruses. (Remove the MEM after washing).
Add 1 ml of MEM containing 2% FBS and 0.4% agarose.
Incubate the cells for 6 days in a cell culture incubator at 35 °C with 5% CO₂.
Count the number of foci expressing AG1 or EGFP using a fluorescence microscope (Figure 3).

Fig. 3 Fluorescence images of Vero cells infected with recombinant viruses.

C. Analysis of the effect of SMase treatment on VLP infection (Figure 1C)

Seed 0.5 ml of a Vero cell suspension at a concentration of 300,000 cells/ml into each well of a 24-well plate.
Incubate the cells in a cell culture incubator at 37 °C with 5% CO₂.
After the cells form a monolayer (24 h post seeding), wash the cells with DMEM once. (Remove the DMEM after washing).
Add 0.1 ml of a RuV-VLP-containing solution at a concentration of 50,000 fluorescent focus-forming units/ml.
Incubate the cells with the RuV-VLP solution at 4 °C (on ice) for 2 h with gentle agitation every 30 min.
Wash the cells with DMEM, which is precooled to 4 °C, twice. (Remove the DMEM after washing).
Add 0.5 ml of DMEM.
Incubate the cells in a cell culture incubator at 37 °C with 5% CO₂.
Immediately thereafter or after 20, 60 or 120 min, replace the culture media with 0.5 ml of DMEM supplemented with 150 mU/ml of SMase.
Incubate the cells in a cell culture incubator at 35 °C with 5% CO₂ for 3 days.
Wash the cells with DPBS once and add 50 μl of lysis buffer. (Remove the DPBS after washing).
Incubate at room temperature for 10 min.
Transfer the cell lysate to a 1.5 ml tube.
Prepare 50 μl of Renilla Luciferase Assay Reagent in a new 1.5 ml tube and add 10 μl of the cell lysate into the Renilla Luciferase Assay Reagent-containing tube.
Mix quickly by flicking the tube with a finger.
Place the tube in a luminometer to measure the luminescence.

Data analysis

Each assay should be conducted in duplicate to calculate the average number of plaques or foci (or average value of luciferase activity). The assays should be repeated at least three times independently to show statistical differences among the samples.

The percent of virus titer (or luciferase activity) for each assay is calculated as follows:

The percent of virus titer (or luciferase activity) (%) = A/B x100

where, A = average number of plaques or foci (or average value of luciferase activity),

B = average number of plaques or foci (or average value of luciferase activity) in the non-treated control.

* For research use only. Not intended for any clinical use.

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Protocol for Analyzing of the Effect of Sphingomyelinase on Rubella Virus Infectivity in Two Cell Lines

Experiment Summary

Materials and Reagents

Equipment

Procedure

Data analysis

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